Method of combatting infectious diseases using dicationic bis-benzimidazoles

ABSTRACT

The present invention provides methods for treating Cryptococcus neoformans and Candida albicans in a subject in need of such treatment. The methods comprises administering to the subject a dicationic bis-benzimidazole in an amount effective to treat the conditions.

The present invention was made with Government support under GrantNumber 1UO1-AI3363 from the National Institutes of Health. TheGovernment has certain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to methods useful in combatting infectiousdiseases. Specifically, this invention relates to methods of combattinginfectious diseases using dicationic bis-benzimidazole compounds.

BACKGROUND OF THE INVENTION

The need for new antifungal agents has become more pronounced because ofthe increase in the number of fungal infections which occur in patientswho are immunocompromised. There is an increased incidence of fungalinfections attributed, for example, to the aggressive use of cancerchemotherapy, organ transplantation, and opportunistic infectionsassociated with acquired immunodeficiency syndrome (AIDS) patients.Fungal infections are among the most common complications of AIDS, aswell as of cancer chemotherapy. The major opportunistic fungal pathogenscausing disseminated mycoses in immunocompromised hosts include Candidaand Cryptococcus.

Currently used antifungal agents for the treatment of systemic mycosescan be classified as polyene antibiotics, including Amphotericin B,flucytosine and synthetic azoles. There can, however, be significantdrawbacks to the use of these agents, including limited efficacy and/ortoxicity. Accordingly, it is an object of the present invention toprovide new compounds useful in the treatment of fungal infections.

SUMMARY OF THE INVENTION

As a first aspect, the present invention provides a method of treatingCryptococcus neoformans in a patient in need of such treatment. Themethod comprises administering to a patient in need of such treatment anamount effective to treat C. neoformans of a compound of Formula I:##STR1## wherein: R₁ and R₂ are each independently selected from thegroup consisting of H, lower alkyl, alkoxyalkyl, cycloalkyl, aryl,alkylaryl, hydroxyalkyl, aminoalkyl, or alkylaminoalkyl, or R₁ and R₂together represent a C₂ to C₁₀ alkyl, hydroxyalkyl, or alkylene or R₁and R₂ together are: ##STR2## wherein n is a number from 1 to 3, and R₁₀is H or --CONHR₁₁ NR₁₅ R₁₆ wherein C₁₁ is lower alkyl and R₁₅ and R₁₆are each independently selected from the group consisting of H and loweralkyl; and

R₃ is H, hydroxy, lower alkyl, cycloalkyl, aryl, alkylaryl, alkoxyalkyl,hydroxycycloalkyl, alkoxycycloalkoxy, hydroxyalkyl, aminoalkyl oralkylaminoalkyl;

A is a heterocyclic aromatic group selected from the group consistingof: ##STR3## wherein R₄, R₅, and R₆ are each independently selected fromthe group consisting of H, lower alkyl, halogen, aryl, arylalkyl,aminoalkyl, aminoaryl, oxyalkyl, oxyaryl, or oxyarylalkyl;

R₁₂ is hydrogen, lower alkyl, hydroxy, aminoalkyl or alkylaminoalkyl, ora physiologically acceptable salt thereof.

In a preferred embodiment of the invention, R₁ and R₂ together representa C₂ to C₄ alkylene, and R₃, R₄, R₅ and R₆ are H and R₁₂ is H or loweralkyl. In one aspect of this embodiment of the invention, R₁ and R₂together represent ##STR4## and R₃ is H.

In another aspect of this embodiment of the invention, R₁ and R₂together represent ##STR5## and R₃ is H.

In yet another preferred embodiment of the invention, each of R₁, R₂,R₃, R₄, R₅ and R₆ are H and R₁₂ is H or lower alkyl.

In yet another preferred embodiment of the invention, each of R₁, R₃,R₄, R₅ and R₆ are H, R₂ is lower alkyl, preferably isopropyl, and R₁₂ isH or lower alkyl.

In yet another preferred embodiment of the invention, A is: ##STR6##wherein R₄, R₅, and R₆ are each H.

In another preferred embodiment of the invention, A is: ##STR7##

wherein R₄ and R₅ are each H and R₁₂ is H or lower alkyl.

In yet another preferred embodiment of the invention, A is: ##STR8##wherein R₄ and R₅ are each H.

As a second aspect, the present invention provides a method of treatingCandida albicans in a patient in need of such treatment. The methodcomprises administering to a patient in need of such treatment acompound of Formula I above in an amount effective to treat C. albicans.

As a third aspect, the present invention also provides a method oftreating a tumor bearing patient in need of such treatment. The methodcomprises administering to a patient in need of such treatment acompound of Formula I above in a therapeutically effective amount.

As a fourth aspect, the present invention provides compounds useful forthe treatment of C. neoformans. The compounds have the structuralFormula (I), described above. Currently preferred compounds of Formula Iinclude, but are not limited to, 2,5-bis(5-amidino-2-benzimidazolyl)pyrrole; 2,5-bis-[5-(2-imidazolinyl)-2-benzimidazolyl] pyrrole;2,6-bis[5-(2-imidazolinyl)-2-benzimidazolyl]pyridine;1-methyl-2,5-bis(5-amidino-2-benzimidazolyl)pyrrole;1-methyl-2,5-bis[5-(2-imidazolyl)-2-benzimidazolyl] pyrrole;1-methyl-2,5-bis[5-(1,4,5,6-tetrahydro-2-pyrimidinyl)-2-benzimidazolyl]pyrrole; 2,6-bis(5-amidino-2-benzimidazoyl)pyridine;2,6-bis[5-(1,4,5,6-tetrahydro-2-pyrimidinyl)-2-benzimidazolyl] pyridine;2,5-bis(5-amidino-2-benzimidazolyl)furan;2,5-bis[5-(2-imidazolinyl)-2-benzimidazolyl]furan;2,5-bis(5-N-isopropylamidino-2-benzimidazolyl)furan; and physiologicallyacceptable salts thereof. Novel compounds useful for treating C.albicans and for combatting tumors are also disclosed.

The foregoing and other objects and aspects of the present invention areexplained in detail in the specification set forth hereinbelow.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "lower alkyl," refers to C1 to C6 linear orbranched alkyl, such as methyl, ethyl, propyl, butyl, isopropyl,sec-butyl, tert-butyl, butyl, pentyl, isopentyl, and hexyl. The term"cycloalkyl" as used herein refers to C3 to C6 cyclic alkyl, such ascyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term "aryl" asused herein refers to C3 to C10 cyclic aromatic groups such as phenyl,naphthyl, and the like, and includes substituted aryl groups such astolyl. The term "hydroxyalkyl" as used herein refers to C1 to C4 linearor branched hydroxy-substituted alkyl, i.e., --CH₂ OH, --(CH₂)₂ OH, etc.The term "aminoalkyl" as used herein refers to C1 to C₄ linear orbranched amino-substituted alkyl, wherein the term "amino" refers to thegroup NR'R'', wherein R' and R'' are independently selected from H orlower alkyl as defined above, i.e., --NH₂, --NHCH₃, --N(CH₃)₂, etc. Theterm "alkoxyalkyl" as used herein refers to C1 to C6 linear or branchedalkoxy, such as methoxy, ethoxy, propyloxy, butyloxy, isopropyloxy, andt-butyloxy.

As noted above, the methods of the present invention are useful fortreating Cryptococcus neoformans. The methods of the present inventionare useful for treating these conditions in that they inhibit the onset,growth, or spread of the condition, cause regression of the condition,cure the condition, or otherwise improve the general well-being of asubject inflicted with, or at risk of contracting the condition.

Subjects to be treated by the methods of the present invention aretypically human subjects although the methods of the present inventionmay be useful with any suitable subject known to those skilled in theart.

As noted above, the present invention provides pharmaceuticalformulations comprising the aforementioned compounds of Formula I, orpharmaceutically acceptable salts thereof, in pharmaceuticallyacceptable carriers for aerosol, oral, and parenteral administration asdiscussed in greater detail below. Also, the present invention providessuch new compounds or salts thereof which have been lyophilized andwhich may be reconstituted to form pharmaceutically acceptableformulations for administration, as by intravenous or intramuscularinjection.

The therapeutically effective dosage of any specific compound, the useof which is in the scope of present invention, will vary somewhat fromcompound to compound, patient to patient, and will depend upon thecondition of the patient and the route of delivery. As a generalproposition, a dosage from about 0.1 to about 50 mg/kg will havetherapeutic efficacy, with still higher dosages potentially beingemployed for oral and/or aerosol administration. Toxicity concerns atthe higher level may restrict intravenous dosages to a lower level suchas up to about 10 mg/kg, all weights being calculated based upon theweight of the active base, including the cases where a salt is employed.Typically a dosage from about 0.5 mg/kg to about 5 mg/kg will beemployed for intravenous or intramuscular administration. A dosage fromabout 10 mg/kg to about 50 mg/kg may be employed for oraladministration. The duration of the treatment is usually once per dayfor a period of two to three weeks or until the Cryptococcus neoformansinfection is essentially controlled. Lower doses given less frequentlycan be used to prevent or reduce the incidence of recurrence of theinfection.

In accordance with the present method, a compound of Formula I, or apharmaceutically acceptable salt thereof, may be administered orally orthrough inhalation as a solid, or may be administered intramuscularly orintravenously as a solution, suspension, or emulsion. Alternatively, thecompound or salt may also be administered by inhalation, intravenouslyor intramuscularly as a liposomal suspension. When administered throughinhalation the compound or salt should be in the form of a plurality ofsolid particles or droplets having a particle size from about 0.5 toabout 5 microns, preferably from about 1 to about 2 microns.

Besides providing a method for treating Cryptococcus neoformans, thecompounds of Formula I also provide a method for prophylaxis againstCryptococcus neoformans in an immunocompromised patient, such as onesuffering from AIDS, who has had at least one episode of Cryptococcusneoformans but who at the time of treatment is not exhibiting signs ofinfection. As Cryptococcus neoformans is an especially potentiallydevastating disease for immunocompromised patients it is preferable toavoid the onset of Cryptococcus neoformans as compared to treating thedisease after it has become symptomatic. Accordingly, the presentinvention provides a method for the prophylaxis against Cryptococcusneoformans comprising administering to the patient a prophylacticallyeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt thereof. The forms for administration of the compound orsalt in accordance with this method may be the same as utilized for thepurpose of actually treating a patient suffering from Cryptococcusneoformans infection.

An additional useful aspect of the present invention is a method forprophylaxis against even an initial episode of Cryptococcus neoformansinfection in an immunocompromised patient who has never experienced anepisode of Cryptococcus neoformans infection. In this respect, a patientwho has been diagnosed as being immunocompromised, such as one sufferingfrom AIDS or ARC (AIDS related complex), even before the onset of aninitial episode of Cryptococcus neoformans infection may avoid or delaysuffering from the infection by having administered a prophylacticallyeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt thereof. The compound or salt may be administered in thesame fashion as in the treatment of patients suffering from Cryptococcusneoformans infection.

The present invention also provides new pharmaceutical compositionssuitable for intravenous or intramuscular injection. The pharmaceuticalcompositions comprise a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, in any pharmaceutically acceptable carrier. Ifa solution is desired, water is the carrier of choice with respect towater-soluble compounds or salts. With respect to the water-insolublecompounds or salts, an organic vehicle, such as glycerol, propyleneglycol, polyethylene glycol, or mixtures thereof, may be suitable. Inthe latter instance, the organic vehicle may contain a substantialamount of water. The solution in either instance may then be sterilizedin any suitable manner, preferably by filtration through a 0.22 micronfilter. Subsequent to sterilization, the solution may be filled intoappropriate receptacles, such as depyrogenated glass vials. Of course,the filling should be done by an aseptic method. Sterilized closures maythen be placed on the vials and, if desired, the vial contents may belyophilized.

In addition to compounds of Formula I or their salts, the pharmaceuticalcompositions may contain other additives, such as pH adjustingadditives. In particular, useful pH adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the compositions may contain microbialpreservatives. Useful microbial preservatives include methylparaben,propylparaben, and benzyl alcohol. The microbial preservative istypically employed when the formulation is placed in a vial designed formultidose use. Of course, as indicated, the pharmaceutical compositionsof the present invention may be lyophilized using techniques well knownin the art.

In yet another aspect of the present invention, there is provided aninjectable, stable, sterile composition comprising a compound of FormulaI, or a salt thereof, in a unit dosage form in a sealed container. Thecompound or salt is provided in the form of a lyophilizate which iscapable of being reconstituted with a suitable pharmaceuticallyacceptable carrier to form a liquid composition suitable for injectionthereof into man. The unit dosage form typically comprises from about 10mg to about 10 grams of the compound or salt. When the compound or saltis substantially water-insoluble, a sufficient amount of emulsifyingagent which is physiologically acceptable may be employed in sufficientquantity to emulsify the compound or salt in an aqueous carrier. Onesuch useful emulsifying agent is phosphatidyl choline.

Other pharmaceutical compositions may be prepared from thewater-insoluble compounds of Formula I, or salts thereof, such asaqueous base emulsions. In such an instance, the composition willcontain a sufficient amount of pharmaceutically acceptable emulsifyingagent to emulsify the desired amount of the compound of Formula I orsalt thereof. Particularly useful emulsifying agents includephosphatidyl cholines, and lecithin.

Further, the present invention provides liposomal formulations of thecompounds of Formula I and salts thereof. The technology for formingliposomal suspensions is well known in the art. When the compound ofFormula I or salt thereof is an aqueous-soluble salt, using conventionalliposome technology, the same may be incorporated into lipid vesicles.In such an instance, due to the water solubility of the compound orsalt, the compound or salt will be substantially entrained within thehydrophilic center or core of the liposomes. The lipid layer employedmay be of any conventional composition and may either containcholesterol or may be cholesterol-free. When the compound or salt ofinterest is water-insoluble, again employing conventional liposomeformation technology, the salt may be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced may be reduced insize, as through the use of standard sonication and homogenizationtechniques.

Of course, the liposomal formulations containing the compounds ofFormula I or salts thereof, may be lyophilized to produce a lyophilizatewhich may be reconstituted with a pharmaceutically acceptable carrier,such as water, to regenerate a liposomal suspension.

Pharmaceutical formulations are also provided which are suitable foradministration as an aerosol, by inhalation. These formulations comprisea solution or suspension of the desired compound of Formula I or a saltthereof or a plurality of solid particles of the compound or salt. Thedesired formulation may be placed in a small chamber and nebulized.Nebulization may be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the compounds or salts. The liquid droplets or solidparticles should have a particle size in the range of about 0.5 to about5 microns. The solid particles can be obtained by processing the solidcompound of Formula I, or a salt thereof, in any appropriate mannerknown in the art, such as by micronization. Most preferably, the size ofthe solid particles or droplets will be from about 1 to about 2 microns.In this respect, commercial nebulizers are available to achieve thispurpose.

Preferably, when the pharmaceutical formulation suitable foradministration as an aerosol is in the form of a liquid, the formulationwill comprise a water-soluble compound of Formula I or a salt thereof,in a carrier which comprises water. A surfactant may be present whichlowers the surface tension of the formulation sufficiently to result inthe formation of droplets within the desired size range when subjectedto nebulization.

As indicated, the present invention provides both water-soluble andwater-insoluble compounds and salts. As used in the presentspecification, the term "water-soluble" is meant to define anycomposition which is soluble in water in an amount of about 50 mg/mL, orgreater. Also, as used in the present specification, the term"water-insoluble" is meant to define any composition which hassolubility in water of less than about 20 mg/mL. For certainapplications, water soluble compounds or salts may be desirable whereasfor other applications water-insoluble compounds or salts likewise maybe desirable.

Examples of compounds exemplary of Formula (I) above include, but arenot limited to:

2,5-bis(5-amidino-2-benzimidazolyl) pyrrole;

2,5-bis-[5-(2-imidazolinyl)-2-benzimidazolyl] pyrrole;

2,6-bis[5-(2-imidazolinyl)-2-benzimidazolyl]pyridine;

1-methyl-2,5-bis(5-amidino-2-benzimidazolyl)pyrrole;

1-methyl-2,5-bis[5-(2-imidazolyl)-2-benzimidazolyl] pyrrole;

1-methyl-2,5-bis[5-(1,4,5,6-tetrahydro-2-pyrimidinyl)-2-benzimidazolyl]pyrrole;

2,6-bis(5-amidino-2-benzimidazoyl)pyridine;

2,6-bis[5-(1,4,5,6-tetrahydro-2-pyrimidinyl)-2-benzimidazolyl] pyridine;

2,5-bis(5-amidino-2-benzimidazolyl)furan;

2,5-bis[5-(2-imidazolinyl)-2-benzimidazolyl]furan;

2,5-bis(5-N-isopropylamidino-2-benzimidazolyl)furan;

and physiologically acceptable salts thereof.

As indicated, the compounds used in the present invention may be presentas pharmaceutically acceptable salts. Such salts include the gluconate,lactate, acetate, tartarate, citrate, phosphate, borate, nitrate,sulfate, and hydrochloride salts.

Methods of combating Candida albicans with the compounds of Formula Iabove are carried out in essentially the same manner as given above, andpharmaceutical formulations of the compounds of Formula I for combatingCandida albicans are prepared in essentially the same manner as givenabove.

The compounds of Formula (I) also show pharmaceutical activity incombatting cancer cells in vitro and may be useful in combattingcorresponding tumors in vivo. For example, the compounds of Formula (I)show cytotoxic activity against leukemia cells. Accordingly, the presentinvention also includes a method of treating a tumor bearing patient inneed of such treatment. The method comprises administering to thepatient a compound of Formula (I) in an amount effective to combat thetumor. Methods of combating tumors with the compounds of Formula I aboveare carried out in essentially the same manner as given above, andpharmaceutical formulations of the compounds of Formula I for combatingtumors are prepared in essentially the same manner as given above.

Further, it is anticipated that the antineoplastic efficacy of thecompounds of Formula (I) can be improved or supplemented by the cojointadministration of these compounds with other known antineoplasticagents, as, for example, in a combination chemotherapy regimen.Exemplary of such known antineoplastic agents are, without limitation,vinca alkaloids such as vincristine, vinblastine, and vindesine;epipodophyllotoxins such as etoposide and teniposide; anthracyclineantibiotics such as daunorubicin, doxorubicin, mitoxantraone, andbisanthrene; actinomycin D; and plicamycin.

The compounds of the present invention may be prepared according tomethods known in the art, particularly in light of the disclosure andexamples set forth below. According to one method, the compounds ofFormula I can be prepared by condensation of an appropriate heterocycliccompound (i.e., pyrrole, pyridine, furan, etc.) dicarboxaldehyde (forexample pyrrole-2,5-dicarboxaldehyde) which can be prepared according tothe teaching of T. Cresp, et al., J.Chem.Soc. Perkins Tran. 1, 2961(1973) with the appropriate diaminophenyl compound (for examplediaminobenzamidine prepared according to T. Fairley, et al., Med.Chem.36, 1746 (1993). The condensation reaction can be conducted according tothe method of S. Kumar, et al., Indian J.Chem 20B, 254 (1981).

The diaminophenyl compounds can be prepared, for example, by reductionof nitro groups of 3,4-dinitrobromobenzene to form3,4-diaminobromobenzene. Nitrilization of this compound to thecorresponding 3,4-diaminonitrilebenzene can be conducted by reactingcopper(I) cyanide with the thus prepared 3,4-dinitrobromobenzene inrefluxing DMF according to the standard techniques. See, J. Spychala, etal., European J. Med. Chem. 29: 363 (1994). The nitrile can then beconverted to the imidate ester by the Pinner methodology, according toB. Das, et al., J. Med. Chem. 20, 1219 (1977). The imidate ester can beconverted into the compounds of Formula (I), for example, by reactionwith ammonium or the appropriate aminoalkane or diaminoalkane (such asethylenediamine, propylenediamine, etc.), to form an amidino group, animidazolinyl group, an 1,4,5,6-tetrahydro-2-pyrimidinyl group,respectively. The bis-nitrile can also be converted to thebis-dicationic compound by fusion of the nitrile directly with thehydrochloride salt of the appropriate diamine by thermolysis. Thistechnique is particularly useful for the preparation of compoundswherein the R₁ and R₂ groups together form a cyclic alkyl.

The compounds of Formula I above can also be prepared by first preparingan appropriate intermediate, such as2,5-bis(5-bromo-2-benzimidazolyl)pyrrole by the base promotedcondensation, for example, of 1-bromo-3,4-diaminobenzene andpyrrole-2,5-dicarboxaldehyde, according to the method of S. Kumar, etal., supra. The intermediate can then be obtained by nitrilizationfollowed by imidate ester formation and conversion into thecorresponding amidino as described above.

The salts of the present invention may be prepared, in general, byreacting two equivalents of the heterocyclic base compound with thedesired acid in solution. After the reaction is complete, the salts arecrystallized from solution by the addition of an appropriate amount ofsolvent in which the salt is insoluble.

The compounds of the present invention are useful not only in methodsfor treating Cryptococcus neoformans and Candida albicans but also inmethods of inhibiting enzymes such as topoisomerase. The compounds ofFormula (I) are particularly useful for inhibiting topoisomerase II.See, S. Doucc-Racy, et al., Proc. Natl. Acad. Sci. USA 83: 7152 (1986).

The present invention will be further illustrated by the followingnon-limiting examples, in which "g" means grams, "mg" means milligrams,"μg" means micrograms, "mmol" means millimoles, "h" means hours, "ml"means milliliter, "M" means molar, "mM" means millimolar, "μM" meansmicromolar, "UV" means ultraviolet, "HCl" means hydrogen chloride, "mp"means melting point, "HCN" means hydrocyanic acid and "°C." meansdegrees Celsius.

For the following examples melting points were recorded using a ThomasHoover (Uni-Melt) capillary melting point apparatus and are uncorrected.¹ H NMR and ¹³ C NMR spectra were recorded employing a Varian GX400spectrometer and chemical shifts (d) are in ppm relative to TMS unlessotherwise noted. Mass spectra were recorded on a VG Instruments 70-SEspectrometer (Georgia Institute of Technology, Atlanta, Ga.). IR spectrawere recorded using a Michelson 100 (Bomem, Inc.) instrument. Elementalanalysis were obtained from Atlantic Microlab Inc. (Norcross, Ga.) andare within +0.4 of the theoretical values. All chemicals and solventswere purchased from Aldrich Chemical Co. or Fisher Scientific.

In the Examples below, the following compound designations are usedthroughout.

    __________________________________________________________________________    Compound #                                                                           Name                                                                   __________________________________________________________________________    1      2,5-bis(5-amidino-2-benzimidazolyl)pyrrole                             2      2,5-bis-[5-(2-imidazolinyl)-2-benzimidazolyl]pyrrole                   3      2,6-bis[5-(2-imidazolinyl)-2-benzimidazolyl]pyridine                   4      1-Methyl-2,5-bis(5-amidino-2-benzimidazolyl)pyrrole                    5      1-methyl-2,5-bis[5-(2-imidazolyl)-2-benzimidazolyl]pyrrole             6      1-Methyl-2,5-bis[5-(1,4,5,6-tetrahydro-2-pyrimidinyl)-2-benzimidazo           lyl]                                                                          pyrrole                                                                7      2,6-bis(5-amidino-2-benzimidazoyl)pyridine                             8      2,6-bis[5-(1,4,5,6-tetrahydro-2-pyrimidinyl)-2-benzimidazolyl]pyrid           ine                                                                    9      2,5-bis(5-amidino-2-benzimidazolyl)furan                               10     2,5-bis[5-(2-imidazolinyl)-2-benzimidazolyl]furan                      11     2,5-bis(5-N-isopropylamidino-2-benzimidazolyl)furan                    __________________________________________________________________________

EXAMPLE 1 Preparation of 2,5-bis(5-amidino-2-benzimidazolyl) pyrrole

2,5-bis(5-amidino-2-benzimidazolyl) pyrrole (Compound 1). A solution ofpyrrole-2,5-dicarboxaldehyde (Cresp, T., Sargent, M., J.Chem.Soc. PerkinTrans. 1, 2961 (1973)) (0.25 g, 2 mmol), 3,4-diaminobenzamidine (FairleyTA, Tidwell RR, Donkor I, Naiman NA, Ohemeng KA, Bentley A and Cory M.J., Med.Chem. 36, 1746 (1993)) (0.6 g, 4 mmol) and 1,4-benzoquinone(0.432 g, 4 mmol) in ethanol (40 ml) was heated at reflux for 4 hours(under nitrogen) (Kumar, S., Konsal, V., Bhaduri, A., Indian J.Chem.20B, 254 (1981). The reaction mixture was cooled to room temperature andthe dark solid was collected by filtration, washed with cold ethanol,anhydrous ether and dried to yield 0.45 g (59%) of the free base. Thissolid was dissolved slowly in hot ethanol (300 ml) and filtered. Thefiltrate volume was reduced to 70 ml and acidified with HCl-saturatedethanol. After standing overnight in the refrigerator, the green solidwas collected by filtration, washed with anhydrous ether and dried undervacuum to yield 0.5 g (76%) yield of solid. mp>300° C. ¹ HNMR (DMSO-d₆)(7.54 (s, 2H, pyrrole), 7.80 (dd, J=8.8 and 0.8 Hz, Ar--H, 2H), 7.87 (d,J=8.4 Hz, 2H, Ar--H), 8.25 (s, 2H, Ar--H), 9.18, 9.48 (brs, brs, NH).Anal. (C₂₀ N₁₇ N₉.3HCl.3H₂ O) C,H,N. MS: m/e 384 (M+1).

EXAMPLE 2 Preparation of 2,5-bis-[5-(2-imidazolinyl)-2-benzimidazolyl]pyrrole

2,5-bis-[5-(2-imidazolinyl)-2-benzimidazolyl] pyrrole (Compound 2). Aprotocol similar to that used in Example 1 above was used for thecondensation of pyrrole-2,5-dicarboxaldehyde and 2-(3,4-diaminophenyl)imidazoline to give a 86% yield of solid. mp>300° C. ¹ HNMR (DMSO-d₆)(4.04 (s, 8H, NCH₂ CH₂ N), 7.39 (s, 2H, pyrrole), 7.86 (d, J=8.8 Hz, 2H,Ar--H), 7.92 (dd, J=8.4 and 1.6 Hz, 2H, Ar--H), 8.44 (s, 2H, Ar--H),10.71 (s, NH). Anal. (C₂₄ H₂₁ N₉.3HCl.4H₂ O) C,H,N. MS: m/e 436 (M+1).

EXAMPLE 3 Preparation of 2,6-bis[5-(2-imidazolinyl)-2-benzimidazolyl]pyridine

2,6-bis[5-(2-imidazolinyl)-2-benzimidazolyl]pyridine (Compound 3). Aprotocol similar to that used in Examples 1 and 2 above was used forcondensation of 2,6-pyridine carboxyaldehyde and2-(3,4-diaminophenyl)imidazoline to give an 85% yield of solid. mp>300°C. ¹ HNMR (DMSO-d₆) (4.05 (s, 8H, N--CH₂ CH₂ N), 7.96 (m, 4H, Ar--H),8.30 (t, 1H, pyridine), 8.49-8.51 (m, 4H, Ar--H), 10.71 (s, NH). Anal.(C₂₅ H₂₁ N₉.3HCl.3H₂ O) C,H,N. MS: m/e 448 (M+1).

EXAMPLE 4 Preparation of1-Methyl-2,5-bis(5-amidino-2-benzimidazolyl)pyrrole

1-Methyl-2,5-bis(5-amidino-2-benzimidazolyl)pyrrole (Compound 4). Aprotocol similar to that described above in Examples 1-3 was employedfor the condensation of 3,4-diaminobenzamidine (Fairley TA, Tidwell RR,Donkor I, Naiman NA, Ohemeng KA, Bentley A and Cory M. J., Med. Chem.36, 1746 (1993) ) with 1-methylpyrrole-2,-5-dicarboxaldehyde to yield0.48 g (46%) of product. mp>300° C.; ¹ HNMR (DMSO-d₆) (4.72 (s, 3H, CH₃--N), 7.33 (s, 2H, pyrrole), 7.73 (dd, J=8 and 1.2 Hz, 2H, Ar--H), 7.80(d, J=8.4 Hz, Ar--H), 8.19 (s, 2H, Ar--H) 9.11, 9.38 (brs, brs,NH-amidine). Anal. (C₂₁ H₁₉ N₉.3HCl.H₂ O) C,H,N. MS: m/e 398 (M+1).

EXAMPLE 5 Preparation of1-methyl-2,5-bis[5-(2-imidazolyl)-2-benzimidazolyl] pyrrole

1-methyl-2,5-bis[5-(2-imidazolyl)-2-benzimidazolyl] pyrrole (Compound5). A protocol similar to that described above in Examples 1-4 wasemployed for the condensation of 2-(3,4-diaminophenyl)-imidazoline with1-methylpyrrole-2,-5 - dicarboxaldehyde. A yield of 83% of solid,mp>300° C., was obtained. ¹ HNMR (4.04 (s, 8H, NCH₂ CH₂ N), 4.72 (s, 3H,CH₃ N), 7.30 (s, 2H, pyrrole), 7.84 (qAB, J=8.4 and 8 Hz, 4H, Ar--H),8.36 (s, 2H, Ar--H), 10.60 (s, NH). Anal. (C₂₅ H₂₃ N₉.3HCl.3H₂ O) C,H,N.MS: m/e 450 (M+1).

EXAMPLE 6 Preparation of1-Methyl-2,5-bis[5-(1,4,5,6-tetrahydro-2-pyrimidinyl)-2-benzimidazolyl]pyrrole

1-Methyl-2,5-bis[5-(1,4,5,6-tetrahydro-2-pyrimidinyl)-2-benzimidazolyl]pyrrole (Compound 6). A protocol similar to that described above forExamples 1-5 was employed for the condensation of2-(3,4-diaminophenyl)tetrahydropyrimidine with1-methylpyrrole-2,5-dicarboxaldehyde. A yield of 83% of solid, mp>300°C., was obtained. ¹ HNMR (2.01 (m, 4H, CH₂), 3.52 (brs. 8H, CH₂ N), 4.72(s, 3H, CH₃ N), 7.31 (s, 2H, pyrrole), 7.60 (d, J=8.4 Hz, 2H, Ar--H),7.80 (d, J=8.4 Hz, 2H, AR-H), 8.06 (s, 2H, Ar--H), 9.99 (s, NH). Anal.(C₂₇ H₂₇ N₉.3HCl.4H₂ O) C,H,N. MS: m/e 478 (M+1).

EXAMPLE 7 Preparation of 2,6-bis(5-amidino-2-benzimidazoyl)pyridine

2,6-bis(5-amidino-2-benzimidazoyl)pyridine (Compound 7). A protocolsimilar to that described in Examples 1-6 above was used to condense2,6-pyridine dicarboxaldehyde with 3,4-diaminobenzamidine to yield 89%of a solid, mp>300° C. ¹ HNMR (DMSO-d₆) (7.79 (dd, J=8.4 and 1.6 Hz, 2H,Ar--H), 7.94 (d, J=8.4 Hz, 2H, Ar--H), 8.28-8.34 (m, 3H, Ar--H),pyridine), 8.51 (d, J=8 Hz, 2H, pyridine), 9.12, 9.45 (brs, brs, NH).Anal. (C₂₁ H₁₇ N₉.3HCl.2H₂ O) C,H,N. MS: m/e 396 (M+1).

EXAMPLE 8 Preparation of2,6-bis[5-(1,4,5,6-tetrahydro-2-pyrimidinyl)-2-benzimidazolyl] pyridine

2,6-bis[5-(1,4,5,6-tetrahydro-2-pyrimidinyl)-2-benzimidazolyl] pyridine(Compound 8). A protocol similar to that described above in Examples 1-7was used to condense 2,6-pyridine dicarboxyaldehyde with2-(3,4-diaminophenyl)tetrahydropyrimidine to give an 89% yield of solid,mp>300° C. ¹ HNMR (DMSO-d₆) (2.03 (m, 4H, CH₂), 3.54 (brs, 8H, CH₂ N),7.66 (d, J=8.4 Hz, 2H, Ar--H), 7.84 (d, J=8.4 Hz, 2H, Ar--H), 8.17 (s,2H, Ar--H), 8.29 (t, 1H, pyridine), 8.43 (d, J=8 Hz, 2H, pyridine),10.04 (s, NH). Anal. (C₂₇ H₂₅ N₉.3HCl.4H₂ O) C,H,N. MS: m/e 476 (M+1).

EXAMPLE 9 Preparation of 2,5-bis(5-amidino-2-benzimidazolyl) furan

2,5-bis(5-amidino-2-benzimidazolyl] furan. A protocol similar to thatdescribed above in Examples 18-25 above was used to condense 2,5-furandicarboxyaldehyde with 3,4-diaminobenzamidine. A solution of 2,5-furandicarboxyaldehyde (0.25 g, 2 mmol), 3,4-diaminobenzamidine (0.6 g, 4mmol) and benzoquinone (0.43 g, 4 mmol) in ethanol (100 mL) was refluxedunder nitrogen for 4 hours. After cooling, solvent was reduced and tothe residue dry ether was added. The precipitated solid was filtered andwashed with dry ether. The yellow-green solid was acidified withconcentrated HCl. After standing overnight ether was added and the solidwas collected by filtration, washed with dry ether, and dried in vacuumat 90° C. for 48 hours. Yield 0.5 g (52.2%) of the yellow-green powder,mp>300° C. MS (FAB): m/z 385 (M⁺ +1); HRMS: calc. mass (free base):385.1525 (M⁺ +1); observed mass: 385.1535, ¹ H NMR (DMSO-d₆, TMS)δ: 9.30s, 4H (N--H); 8.95 s, 4H (N--H); 8.19 s, 2H (phenyl); 7.81 d, 2H, J=8.8Hz; 7.72 d, 2H, J=8.4 Hz; 7.60 s 2H (furan), ¹³ C NMR (DMSO-d₆ +D₂ O)δ:166.8; 146.3; 146.1; 142.2; 139.7; 123.4; 122.7; 117.1; 116.1; 115.4.Anal. (C₂₀ H₁₆ N₈ O.2HCl.1.5H₂ O) C,H,N

EXAMPLE 10 Preparation of 2,5-bis[5-(2-imidazolinyl)-2-benzimidazolyl]furan

2,5-bis[5-(2-imidazolinyl)-2-benzimidazolyl] furan. A protocol similarto that described above in Examples 18-26 above was used to condense2,5-furan dicarboxyaldehyde with 2-(3,4-diaminophenyl) imidazoline. Asolution of 2,5-furan dicarboxyaldehyde (0.25 g, 2 mmol),2-(3,4-diaminophenyl)imidazoline (0.7 g, 4 mmol) and benzoquinone (0.43g, 4 mmol) in ethanol (100 mL) was refluxed under nitrogen for 4 hours.After cooling, solvent was reduced and to the residue dry ether wasadded. The precipitated solid was filtered and washed with dry ether.The yellow-green solid was acidified with concentrated HCl. Afterstanding overnight ether was added and solid was collected byfiltration, washed with dry ether, and dried in vacuum at 90° C. for 3days. Yield 0.45 g (38.1%) of the green powder, mp>300° C. MS (FAB): m/z437 (M⁺ +1); HRMS: calc. mass (free base): 437.1838 (M⁺ +1); observedmass: 437.1832, ¹ H NMR (DMSO-d₆, TMS)δ: 10.53 s, 4H (N--H); 8.38 s, 2H;7.87 d, 2H, J=8.5 Hz; 7.83 d, 2H, J=8.2 Hz; 7.62 s, 2H; 4.04 s, 8H. ¹³ CNMR (DMSO-d₆ +D₂ O, TMS): δ 166.3; 146.2; 146.1; 142.3; 139.8; 123.7;117.6; 116.9; 116.1; 115.5; 45.0. Anal. (C₂₄ H₂₀ N₈ O.2HCl.5H₂ O) C,H,N

EXAMPLE 11 Preparation of 2,5-bis(5-N-isopropylamidino-2-benzimidazolyl)furan

2,5-bis(5-N-isopropylamidino-2-benzimidazolyl] furan. A protocol similarto that described above in Examples 18-27 above was used to condense2,5-furan dicarboxyaldehyde with 3,4-diamino-N-isopropylbenzamidine. Asolution of 2,5-furan dicarboxyaldehyde (0.25, 2 mmol),3,4-diamino-N-isopropylbenzamidine (0.77 g, 4 mmol), and benzoquinone(0.43 g, 4 mmol) in ethanol (100 mL) was refluxed under nitrogen for 4hours. After cooling, solvent was reduced and to the residue dry etherwas added. The precipitated solid was filtered and washed with dry etherand dried. After drying the green solid was dissolved in anhydrousethanol saturated with HCl (50 mL) and heated until boiling started,then allowed to cool. The green solid was collected by filtration, anddried in vacuum at 90° C. for 3 days. Yield 0.67 g (53.6%) of theyellow-green powder, mp>300° C. MS (FAB): m/z 469 (M⁺ +1); HRMS: calc.mass (free base): 469.2464 (M⁺ +1); observed mass: 469.2475, ¹ H NMR(DMSO-d₆, TMS): δ 9.60+9.58 s+s, 2H (N--H); 9.45 s, 2H (N--H); 9.45 s 2H(N--H); 9.04 s, 2H (N--H); 8.06 s, 2H (phenyl); 7.82 d, 2H, J=8.4 Hz;7.69 s, 2H (furan); 7.62 d, 2H, J=8.2 Hz; 4.09 m, 2H (CH), J=7.02 Hz;1.32 d 12H (CH₃), J=6.3 Hz; ¹³ C NMR (DMSO-d₆ +D₂ O, TMS): δ 162.8;145.9; 145.1; 140.9; 138.5; 124.5; 124.0; 116.9; 115.9; 115.9; 45.9;21.7. Anal. (C₂₆ H₂₈ N₈ O.3HCl.5H₂ O) C,H,N

EXAMPLE 12 Activity of Compounds Against Cryptococcus neoformans andCandida albicans

The activity of various compounds of Formula I against Cryptococcusneoformans and Candida albicans is shown in Table 1. Activity of thecompounds were assessed using a standard in vitro fungal cell growthinhibition assay (the broth dilution antifungal susceptibility testingof yeast, proposed standard document M27-P, validated by the NationalCommittee for Clinical Laboratory Standards, 1992). Briefly, this brothdilution procedure uses RPMI media, and an inoculum of 10⁴ cells.Control tubes contained media alone. After the minimal inhibitorconcentration was determined by the above procedure, tubes with novisible growth were subcultured to determine the minimum fungicidalconcentration by using criteria of less than 0.01% survival of originalinoculum. Two organisms were tested: (1) H99, a clinical isolate of C.neoformans which is fully susceptible to azoles and polyenes in vitroand in vivo; and (2) A39, a clinical isolate of C. albicans which isfully susceptible to azoles and polyenes.

The MFC's (minimum fungicidal concentration) against these two importantAIDS-associated fungal infections are shown in Table 1. MFCs weredetermined following the method of McGinnis.

                                      TABLE 1                                     __________________________________________________________________________                     MFC (μM)      C. neoformans                                                                           ΔT.sub.m                                                                    L1210                                          C.    C.   IC.sub.50 (μM)                                                                   topoisomerase (μM)                                                                   oligo                                                                             Cells                         Compound                                                                            A       Y  neoformans                                                                          albicans                                                                           S. cerevisiae                                                                       I    II   binding                                                                           IC.sub.50                     __________________________________________________________________________                                                    (μM)                       1     pyrrole Am 2.85  5.71 45    50-100                                                                             >5   18.1                                                                              0.21                          2     pyrrole Im 107   26.8 <1    >100 <6.25                                                                              17.6                                                                              1.07                          3     pyridine                                                                              Im 5.2   2.6  10    250-500                                                                            6.25 15.8                                                                              1.26                          4     1-methylpyrrole                                                                       Am 5.69  2.84 90    5-6  1-2  20.3                                                                              0.81                          5     1-methylpyrrole                                                                       Im 2.54  5.09 >100  25-50                                                                              1.25 22.5                                                                              1.51                          7     pyridine                                                                              Am >179  >179 95    125-250                                                                            <62  15.3                                                                              13.64                         8     pyridine                                                                              THP                                                                              19.02 >52  304   250-500                                                                            50-60                                                                              14.8                                                                              3.0                           9     furan   Am 1.61  6.44                                                   10    furan   Im 2.60  5.21                                                   __________________________________________________________________________     Notes                                                                         ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                

EXAMPLE 13 Nucleic Acid Binding Measurements

DNA binding potency was determined for each of the compounds becauseprevious work had determined that the binding of dicationic molecules toDNA is a prerequisite for their antimicrobial activity. See e.g.,Tidwell, R. R., et al., Antimicrob. Agents Chemother. 37, 1713-1716(1993); Bell, C. A. , et al. , Antimicrob. Agents Chemother. 35,1099-1107 (1991). The DNA binding of the compounds was determined by thechange in melting of the DNA bound to the compounds. The method is welldocumented and is a considered a standard method for determining DNAbinding strength. See Cory, M.. et al., J. Med. Chem. 25, 431-438(1992). In brief, a UV-visible light spectrophotometer with a cuvettechanger was interfaced to a microcomputer that recorded the cuvettetemperature and DNA-absorbance data at 259 nm as the sample was heatedat a rate of 18° C./h. Calf thymus DNA was used at an initial absorbanceof 0.3 A₂₅₉. The midpoint of each denaturation curve was determinedafter graphic selection on the computer of the starting and endingabsorbance temperature for each curve of each experiment. DNA or DNAbound to experimental compound was run in each experiment and the ΔT_(m)s were determined from the polynucleotide T_(m) for that experiment. Thegreater the change in melting point, the more potent the DNA binding ofthe molecules. The results are set forth in Table 1 above.

EXAMPLE 14 Anti-tumor Activity

Source: L1210 cells were obtained from the American Type CultureCollection (ATCC#CCL219). This line is a mouse lymphocytic leukemia linefirst described by E. Law et al., J. Natl. Cancer Inst. 10, 179-192(1949) as a tumor arising in a mouse following skin paintings with 0.2%methycholanthrene. The first report of suspension culture was reportedby G. Moore et al. J. Natl. Cancer Inst. 36, 405-421 (1966). This linehas been used extensively for routine screening programs of chemicalagents and natural products for cytotoxic activity and is also utilizedin preliminary testing for antitumor activity by the NCI in cancerchemotherapy screening studies.

The in vitro cytotoxicity studies were performed essentially asdescribed by Denizot and Lang, "Rapid colorimetric assay for cell growthand survival: Modification of tetrazolium dye procedure giving improvedsensitivity and reliability", J. Immunological Methods 89, 271 (1986),but with the following modifications. Since many of the drugs had colorinterference, the viability of the cells was determined with a ³H-thymidine incorporation method. L1210 cells in DMEM+10% fetal calfserum were plated at 2.0×10⁴ cells/well. An equal volume of testcompound was added to each well diluted to 2× the final concentration inthe above medium. Final drug concentrations ranged from 0.05 to 50 μM.After 24 hours at 37° C., 2.5 μCi of ³ H-Thymidine (5 Ci/mmol) was addedto each well. At 48 hours, the cells were harvested onto glass filtersand radioactivity was measured with a scintillation counter.

The IC₅₀ value was determined as the concentration leading to a 50%reduction of ³ H-thymidine incorporation relative to control wells. Eachvalue shown in Table 1, above, resulted from 6 replicates for eachcompound.

The foregoing examples are illustrative of the present invention and arenot intended to be construed as limiting thereof. The invention isdefined by the following claims, with equivalents of the claims to beincluded therein.

That which is claimed is:
 1. A method of treating Cryptococcusneoformans in a subject in need of such treatment, comprisingadministering to said subject a compound of Formula I-A: ##STR17##wherein: R₁ and R₂ are each independently selected from the groupconsisting of H, lower alkyl, alkoxyalkyl, cycloalkyl, aryl, alkylaryl,hydroxyalkyl, aminoalkyl, or alkylaminoalkyl, or R₁ and R₂ togetherrepresent alkylene or R₁ and R₂ together are: ##STR18## wherein n is anumber from 1 to 3, and R₁₀ is H or --CONHR₁₁ NR₁₅ R₁₆ wherein R₁₁ islower alkyl and R₁₅ and R₁₆ are each independently selected from thegroup consisting of H and lower alkyl; andR₃ is H, hydroxy, lower alkyl,cycloalkyl, aryl, alkylaryl, alkoxyalkyl, hydroxycycloalkyl,alkoxycycloalkoxy, hydroxyalkyl, aminoalkyl or alkylaminoalkyl; A₁ is--N(R₁₂)--; wherein R₁₂ is hydrogen, lower alkyl, hydroxy, aminoalkyl oralkylaminoalkyl; and R₄ and R₅ are each independently selected from thegroup consisting of H, lower alkyl, halogen, aryl, arylalkyl,aminoalkyl, aminoaryl, oxyalkyl, oxyaryl, or oxyarylalkyl, or aphysiologically acceptable salt thereof, in an amount effective to treatCryptococcus neoformans.
 2. The method according to claim 1, whereinsaid subject is afflicted with Cryptococcus neoformans.
 3. The methodaccording to claim 1, wherein said subject is at risk of developingCryptococcus neoformans and said compound is administered in aprophylactically effective amount.
 4. The method according to claim 1,wherein R₁ and R₂ together represent a C₂ to C₄ alkylene, and R₃ is H.5. The method according to claim 4, wherein R₁ and R₂ together representC₂ alkylene.
 6. The method according to claim 4, wherein R₁ and R₂together represent C₃ alkylene.
 7. The method according to claim 1,wherein each of R₁, R₂ and R₃ is H.
 8. The method according to claim 1,wherein each of R₁ and R₃ is H and R₂ is lower alkyl.
 9. The methodaccording to claim 1, wherein said compound is selected from the groupconsisting of:2,5-bis(5-amidino-2-benzimidazolyl)pyrrole;2,5-bis-[5-(2-imidazolinyl)-2-benzimidazolyl]pyrrole;1-methyl-2,5-bis(5-amidino-2-benzimidazolyl)pyrrole;1-methyl-2,5-bis[5-(2-imidazolyl)-2-benzimidazolyl]pyrrole and1-methyl-2,5-bis[5-(1,4,5,6-tetrahydro-2-pyrimidinyl)-2-benzimidazolyl]pyrrole;or a physiologically acceptable salt thereof.